Compositions and method for preventing reactogenicity associated with administration of immunogenic live rotavirus compositions

ABSTRACT

The present invention provides compositions for making a medicament and methods for the administration of a vaccine compositions for protection against human rotaviral disease without significant reactogenicity. Human x rhesus reassortant rotavirus compositions were made which when administered during the first 7 to about 10 days of life, provided a composition which was non-reactogenic followed by booster immunizations at 16 to 18 weeks or 14 to 20 weeks, up to 1 year of age. The immune response induced by the initial neonatal administration of the live rotavirus vaccine composition protects the infant from the reactogenicity of the composition when administered as a second vaccine dose at or after 2 months of age. Administration of the immunogenic composition also is expected to ablate or significantly diminish the increase in the excess of intussusception observed 3 to 7 days following administration of the initial dose of rotavirus vaccine at about 2 to 4 months.

BACKGROUND OF THE INVENTION

Rotaviruses are a major cause of acute dehydrating diarrhea in infantsand young children. Rotavirus disease accounts for 25% to 30% ofgastroenteritis deaths in infants and young children in developingcountries and approximately 50,000-100,000 hospitalizations of childrenyounger than five years of age in the United States. In developingcountries, it has been estimated that over 870,000 infants and youngchildren less than 5 years of age die from rotavirus disease annually.For this reason, a safe effective vaccine is needed to prevent severerotavirus disease in infants and young children.

A primary strategy for rotavirus vaccine development has been based on a“Jennerian” approach, which takes advantage of the antigenic relatednessof human and animal rotaviruses and the diminished virulence of animalrotavirus strains in humans. Kapikian et al., in Vaccines, Chanock etal., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp. 151-159 (1987). Several candidate live oral rotavirus vaccineshave been developed using this approach, where an antigenically-relatedlive virus derived from a nonhuman host is used as a vaccine forimmunization against its antigenically related human virus counterpart.Examples of animal rotaviruses that have been used to vaccinate humansinclude bovine rotavirus strain NCDV (RIT4237, Vesikari et al., Lancet,2:870-811 (1983)), bovine rotavirus strain UK (Wyatt et al. PAHOCopublication series no. 1, pp 17-28 (1985), bovine rotavirus strain WC3(Clark et al., Am. J. Dis. Child., 140:350-356 (1986)) and rhesus monkeyrotavirus (RRV) strain MMU 18006 (U.S. Pat. No. 4,571,385, Kapikian etal., in Vaccine 85, Lerner et al., eds., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., pp. 357-367 (1985)).

The protective efficacy among different monovalent bovine and monovalentsimian rotavirus vaccines has proved to be variable (Vesikari, in ViralInfections of the Gastrointestinal Tract, Kapikian, ed., Marcel Dekker,Inc., pp. 419-442 (1994)); Kapikian ibid. pp. 443-470 (1994). Also, highconcentrations of bovine rotavirus have been required to produce asatisfactory immune response in humans (10⁷-10⁸ plaque forming units(pfu)) (Vesikari et al., Ped. Inf. Dis. 4:622-625 (1985); Bernstein etal., J. Infect. Dis. 162:1055-1062 (1990)). The variable efficacy ofthese compositions can in part be attributed to the fact that the targetpopulation of two- to five-month old infants characteristicallydeveloped a homotypic immune response following vaccination (Kapikian etal., Adv. Exp. Med. Biol, 327:59-69 (1992); Bernstein et al., J. Infect.Dis., 162:1055-1062 (1990); Green et al., J. Infect. Dis., 161:667-679(1990); and Vesikari, Vaccine, 11:255-261 (1993)).

Clinically relevant human rotaviruses are members of the Group Arotaviruses. These viruses share a common group antigen mediated by VP6,a protein located on the virus intermediate shell. Also, serotypespecificity depends on the presence of the VP4 (protease sensitive or Ptype) and VP7 (glycoprotein or G type) proteins located on the virusouter shell (also often referred to as the virus capsid), both of whichindependently induce neutralizing antibodies (Kapikian et al., inVirology, Fields et al., eds., Raven Press, New York, N.Y., pps.1353-1404 (1995)).

Group A rotaviruses that infect humans have been classified into tendistinct VP7 serotypes by neutralization assays. Amino acid sequenceanalysis has indicated that within each serotype amino acid identitywithin two major variable regions was high (85-100%); however, aminoacid identity between strains of different serotypes was significantlyless (Green et al., Virology 168:429-433 (1989); Green et al., Virology161:153-159 (1987); and Green et al., J. Virol. 62:1819-1823 (1988)).Concordance between relationships among rotaviruses as determined byvirus neutralization assay or sequence analysis of VP7 has beendemonstrated. Therefore, a reference strain can be routinely used inclinical studies as a representative of rotavirus strains within itsserotype.

To achieve protection against each of the four epidemiologically andclinically most important G serotypes (VP7) (numbered 1, 2, 3, and 4),the Jennerian approach has been modified by the production ofreassortant rotaviruses. Reassortant rotavirus strains were constructedby coinfecting tissue culture cells with a rotavirus of animal origin(i.e., rhesus or bovine rotavirus) and a human rotavirus strain.Reassortant viruses produced during coinfection that contained a singlehuman rotavirus gene encoding VP7 from the human strain and the 10remaining rotavirus genes from the animal strain were selected byexposing the progeny of the coinfection to a set of monoclonalantibodies directed to the VP7 of the animal strain. (See, for example,U.S. Pat. No. 4,571,385; Midthun et al., J. Clin. Microbiol. 24:822-826(1986); and Midthun et al., J. Virol. 53:949-954 (1985), incorporatedherein by reference).

Studies of human×rhesus rotavirus reassortants and human×bovinerotavirus reassortants containing the VP7 gene from a human strain havedemonstrated that the VP4 neutralization protein of the animal rotavirusparent dominates the immune response in infants vaccinated with thesehuman×animal rotavirus reassortants. This probably reflects the absenceof animal rotavirus VP4 antibodies among the antibodies transferredtransplacentally from the mother to the infant in utero. Nevertheless,the immune response to human rotavirus VP7 that is partially blunted bymaternally derived VP7 antibodies is sufficient to provide protectionand thus VP7 antibodies form the basis of the modified Jennerianapproach (Flores et al., J. Clin. Microbiol. 27:512-518 (1989);Perez-Schael et al., J. Clin. Microbiol. 28:553-558 (1990); Flores etal., J. Clin. Microbiol. 31:2439-2445 (1993); Christy et al., J. Infect.Dis. 168:1598-1599 (1993); Clark et al., Vaccine 8:327-332 (1990);Treanor et al., Pediatr. Infect. Dis. J. 14:301-307 (1995); Madore etal., J. Infect. Dis. 166:235-243 (1992); and Clark et al., J. Infect.Dis. 161:1099-1104 (1990).

In studies using a single rhesus rotavirus reassortant bearing a singlehuman rotavirus gene, namely the gene that encodes VP7, it was observedthat the protective immunological response of such a reassortant wascharacteristically homotypic in infants less than six months of age(Green et al., J. Infect. Dis. 161:667-679 (1990)). This observationprovided further evidence for the importance of VP7-associated immunityin immunization against rotavirus disease.

Multivalent rotavirus vaccine compositions have been developed. Inparticular, three human×rhesus rotavirus reassortants representing humanserotypes 1, 2 and 4 have been combined with a rhesus rotavirus strain(RRV) (the latter sharing neutralization specificity with human serotype3) to form a quadrivalent vaccine composition (RRV-TV)(Perez-Schael etal., J. Clin. Microbiol. 28:553-558 (1990), Flores et al., J. Clin.Microbiol. 31:2439-2445 (1993), each incorporated herein by reference).As with the monovalent rhesus rotavirus, the human×rhesus reassortantrotavirus vaccine compositions were found to characteristically producea transient low level febrile condition in approximately 15% to 33% ofthe infants vaccinated (Perez-Schael et al., supra; Bernstein et al.,JAMA 273:1191-1196 (1995); Flores et al., Lancet 336:330-334 (1995);Flores et al., J. Clin. Microbiol. 31:2439-2445 (1993); Halsey et al.,J. Infect Dis. 158:1261-1267 (1988); Taniguichi et al., J. Clin.Microbiol. 29:483-487 (1991); Simasathien et al., Pediatr. Infect. Dis.J. 13:590-596 (1994); Madore et al., J. Infect. Dis. 166:235-243 (1992);and Joensuu et al., Lancet 350:1205-1209 (1997). This transient febrileepisode or condition, although generally considered acceptable by theparents and health care providers of the clinical trial, could possiblybe a deterrent in certain situations, such as, in premature infants whomay have low levels of passively acquired maternal antibodies torotavirus and the like.

Results of studies in humans with bovine rotavirus strains NCDV, WC3 andUK (VP7 serotype 6) indicate that these particular bovine rotavirusstrains cause significantly fewer febrile reactions than the rhesusrotavirus-based vaccines. Further, bovine rotavirus was not found to beas immunogenic as the rhesus rotavirus when administered to humans. Thebovine rotavirus strain NCDV (RIT4237 vaccine) has also been evaluatedin neonates (Vesikari et al. Pediatr. Infect. Dis. J. 6:164-169 (1987)).In these trials, the bovine RIT4237 vaccine or placebo was administeredto 244 newborn infants orally at a dose of 10^(8.3) tissue cultureinfectious doses 50 (TCID₅₀). This high dosage of the vaccinecomposition required to achieve infection indicated that this virus wasover attenuated. The initial vaccination was not followed by a seconddose. Another study was conducted with the same overattenuated vaccinecomposition, RIT 4237, wherein the composition was given to newborns andat 7 months of age. No vaccine associated reactions were observed(Vesikari et al., Acta Paediatr. Scand. 80:173-180 (1991)).

Similar studies have been conducted in newborns with a rhesus rotaviruscomposition (MMU-18006) as a single dose administration. No reactionscould be attributed to administration of this vaccine composition.Serologic responses to the composition were not observed by complementfixation, neutralization or a rhesus rotavirus epitope-specificcompetition assay. The safety and immunogenicity of an oral tetravalenthuman×rhesus reassortant rotavirus vaccine (RRV-TV) was tested innewborns with the addition of a second dose at the age of 6 to 8 weeksto determine if immunogenicity could be enhanced (Dagan et al., Pediatr.Infect. Dis. J. 11:991-996 (1992)). In this study however, a positivecontrol group of older infants, 2 to 6 months, who would be expected todevelop a febrile response to vaccine with a frequency of about 15 to30%, was not studied simultaneously. For this reason, the ability ofneonatal vaccination to prevent or ablate reactogenicity associated withadministration of the second dose of vaccine at 2 months of age, orlater, could not be established.

The RRV-TV vaccine was licensed by the Federal Drug Administration onAug. 31, 1998, for oral administration to infants at two (2), four (4),and six (6) months of age. It is estimated that one million doses havebeen administered since licensure. Post licensure surveillance detectedan apparent excess in the reported incidence of intussusception ininfants predominantly at vaccination. It is considered that thisclustering of cases is associated with the administration of the newrotavirus composition. Although the cause of intussusception is notknown several possible mechanisms for how it may occur followingadministration of the vaccine have been suggested, including enlargementof lymphoid tissue (Peyer's patches) along the gastrointestinal tractthat might lead them to invaginate and serve as a lead point forintussusception.

With the previously licensed rotavirus composition removed from themarket and the important level of protection in humans against rotavirusinfection provided by vaccination, a composition or medicament fortreatment or prevention of rotaviruses infection which protects againstor ablates the relatively high reactogenicity of live rotavirus vaccinecompositions, including the human×rhesus multivalent composition, asmanifested by a transient low level febrile condition that may alsoreduce or eliminate intussusception in infants after immunization.Surprisingly, the present invention fulfills these and other relatedneeds.

SUMMARY OF THE INVENTION

The present invention provides for the use of an immunogenic liverotavirus composition for the manufacture of a medicament for thetreatment or prevention of a rotavirus infection without causingtransient low level fever or causing intussusception, said treatment orprevention comprising administering a safe and effective amount of theimmunogenic live rotavirus composition to an infant within about sevento about ten days of life followed by at least one additionaladministration of a safe and effective amount of the immunogenic liverotavirus composition prior to six months of age.

Compositions useful in manufacturing medicaments of the presentinvention can comprise a rhesus rotavirus immunologically cross-reactivewith human rotavirus serotype 3. Additionally, human×rhesus rotaviruscan be useful in preparation of the live immunogenic composition.Typically, the composition comprises a plurality of human×rhesusreassortant rotaviruses of different serotypes which are clinicallyrelevant. Such a multivalent human×rhesus rotavirus composition can alsobe manufactured to contain a rhesus rotavirus immunologicallycross-reactive with human rotavirus serotype 3. One particularly usefulcomposition comprises a human×rhesus reassortant rotavirus of humanrotavirus serotype 1, a human×rhesus reassortant rotavirus of humanrotavirus serotype 2, a human×rhesus reassortant rotavirus of humanrotavirus serotype 4, and a rhesus rotavirus immunologicallynon-reactive with human rotavirus serotype 3.

In another embodiment of the present invention, plurality of ahuman×bovine reassortant rotavirus can be useful in the manufacture ofthe immunogenic live rotavirus composition for the treatment andprevention of rotarvirus infection without causing transient low levellevel or causing intussusception. The compositions can comprise aplurality of human×bovine reassortant rotaviruses of different serotypeswhich are clinically relevant. Particularly useful are compositionswhich comprise a human×bovine reassortant of human rotavirus serotype 1,a human×bovine reassortant of human rotavirus serotype 2, a human×bovinereassortant of human rotavirus serotype 3, and a human×bovinereassortant rotavirus of human rotavirus serotype 4. The compositionscan further comprise human×bovine reassortant rotavirus including, forexample, those of human rotavirus serotypes 5 and/or 9, or abovine×bovine reassortant rotavirus with human rotavirus VP7 serotype 10specificity, or a human rotavirus serotype VP4 1AX bovine rotavirus UKreassortant.

Immunogenic compositions can be formulated as a medicament wherein eachrotavirus or reassortant rotavirus is administered separately or invarious compositions. Typically, the medicament is formulated as acombined composition comprising a plurality or rotavirus and/orreassortant rotavirus of a human rotavirus serotype. The medicamentcomprising the immunogenic live rotavirus can be formulated foradministration to the alimentary tract of an individual. Typically, aliquid suspension is used for administration of the medicament of thepresent invention.

For the treatment or prevention of rotavirus without causing transientlow level fever and without causing intussusception, the medicamentcomprising a safe and effective amount of the immunogenic live rotaviruscompositions is administered within about seven to about ten days oflife followed by at least one additional administration of a safe andeffective amount of the medicament comprising an immunogenic liverotavirus composition prior to six months of age. Typically, the secondadministration of the medicament of the present invention is at about 6to about 10 weeks of age or at about 14 to about 18 weeks of age. Asubsequent administration of the medicament is at about 14 to 18 weeksof age or about 22 to about 26 weeks of age respectively.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides compositions and methods useful forpreventing reactogenicity associated with the administration ofimmunogenic live rotavirus compositions in humans. The compositionsuseful in the manufacture of a medicament for the prevention andtreatment of rotavirus infection without causing transient low levelfever typically seen when infants received their first dose of therotavirus composition between about 2 to 6 months of age and further, toreduce the risk of intussusception in infants 3 to 7 days following thefirst dose of the rotavirus composition.

As used herein, “reactogenic” or reactogenicity denotes a mild transientfever occurring during the week following administration of theimmunogenic composition. A fever is defined in the context of thepresent invention as the development of a rectal temperature of greaterthan or equal to 38.1° C. in a pediatric vaccinee.

The compositions of the present invention elicit the production of animmune response that is at least partially protective against symptomsof serious rotaviral disease, such as severe diarrhea and dehydration,when the individual is subsequently infected with a wild-type humanrotavirus strain. As the reasserted viruses of the immunogeniccomposition infect the host alimentary tract, typically the immunogeniccomposition of the present invention will not cause clinically relevantfever or reaction in the vaccinee. Following vaccination, there aredetectable levels of host engendered serum antibodies which are capableof neutralizing the serotypes of rotavirus that make up the immunogeniccomposition. In particular, the multivalent immunogenic composition ofthe present invention will produce an immunological response to most, ifnot all, of the clinically relevant group A human rotaviruses prevalentin different settings. The teachings of the present invention are notlimited to those human rotavirus serotypes currently recognized ofclinically relevant, but also include those serotypes of human rotavirusthat may emerge as clinically relevant in the future.

The reassorted rotavirus which is a component of the multivalentimmunogenic composition used in the methods of the present invention isadministered in an isolated and typically purified form. By isolated ismeant to refer to reassorted rotavirus that has been separated fromother cellular and viral products of its manufacture, such as wild typevirus and other heterologous components of a cell culture or othersystems.

Generally, rotavirus reassortants are produced by coinfection ofmammalian cells in culture with a tissue culture-adapted animalrotavirus, i.e., bovine, rhesus, and the like, and a tissueculture-adapted human rotavirus. Typically, African green monkey kidney(AGMK) cells are used as the host cells for co-infection. Followingco-infection with the animal and human rotavirus strains, selection ofthe desired reassortant is typically achieved by exposing the growthyield of co-infected cultures to neutralizing antibodies specific forthe protein product of the animal rotavirus gene that is to be replacedby the human rotavirus gene (See, U.S. Pat. No. 4,571,385, incorporatedherein by reference). In particular, polyclonal serum or monoclonalantibody specific for rhesus or bovine rotavirus VP7 and/or VP4 proteinscan be used. After several rounds of plaque purification and subculture,selected reassortants are characterized for serotype and genotype.Serotype is typically determined by plaque reduction neutralization(PRN) assay or enzyme immunoassay. Genotype is typically determined bygel electrophoresis and RNA-RNA hybridization of the viral genome.Rotavirus reassortants having only the human VP7 or VP4 gene aretypically selected in the present multivalent immunogenic compositions.Reassortants comprising multiple human rotavirus genes can also be used.In this regard, reassortant rotaviruses of interest are particularlythose encoding the human rotavirus VP7 and/or the human rotavirus VP4gene products.

In the present invention, particularly preferred rotavirus reassortantsare human rotavirus and rhesus rotavirus reassortants comprising thehuman rotavirus gene encoding VP7 and the remaining ten rotavirus genesof rhesus rotavirus origin. Other animal rotavirus strains can also beused to make reassortant rotavirus as long as the compositions arecapable such as bovine UK rotavirus of inducing a serologic response ina vaccinee when administered at a dosage of about 10^(6.0) plaqueforming units for each rotavirus serotype.

Propagation of the reassorted rotavirus can be in a number of cellcultures which support rotavirus growth. Preferred cell cultures forpropagation of rotavirus reassortants for vaccine use include primary orsecondary simian African green monkey kidney cells (AGMK), qualifieddiploid simian FRhL-2 cells and qualified simian heteroploid Vero cells.Cells are typically inoculated with rotavirus reassortants at amultiplicity of infection ranging from about 0.1 to 1.0 per cell, ormore, and are cultivated under conditions appropriate for viralreplication, for about 3-5 days, or as long as necessary for virus toreach an adequate titer. Rotavirus reassortants are harvested frominfected cell culture and separated from cellular components, typicallyby well known clarification procedures, e.g., centrifugation, and may bepurified as desired using procedures well known to those skilled in theart.

In a preferred embodiment for use as an immunogenic composition, ahuman×rhesus reassortant rotavirus of VP7 serotype 1, serotype 2, andserotype 4, and a rhesus rotavirus cross-reactive with human rotavirusserotype 3 are used as a quadrivalent vaccine. Typically, theimmunogenic composition will be admixed to form a combined compositionfor simultaneous administration. The final ratio of each rotavirusserotype is determined by the immunogenicity of the individual rotavirusreassortants. Although not preferred, each rotavirus or reassortantrotavirus, can also be administered in a sequential manner to provide aneffective vaccine formulation.

The immunogenic composition may be introduced into a host, particularlyhumans, with a physiologically acceptable carrier and/or adjuvant.Useful carriers include, e.g., citrate-bicarbonate buffer, bufferedwater, normal saline, and the like. The resulting aqueous solutions maybe packaged for use as is, or lyophilized, as desired, usinglyophilization protocols well known to the artisan. Lyophilized viruswill typically be maintained at about 4° C. When ready for use thelyophilized preparation is combined with a sterile solution prior toadministration, as mentioned above.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, tri-ethanolamine oleate,citrate-bicarbonate, or the like. When the composition is administeredorally it may also be necessary to provide the individual a buffersolution to partially neutralize stomach acid and protect thereassortant rotavirus while passing to the intestine. Buffer solutionsappropriate for this use include sodium bicarbonate, citratebicarbonate, or the like. Upon immunization with a human×bovinereassortant rotavirus composition of the present invention, particularlyvia the oral route, the immune system of the host responds to thecomposition by producing both local secretory and serum antibodiesspecific for the rotavirus proteins. As a result of the administrationof the composition, the host becomes at least partially or completelyimmune to human rotavirus disease caused by a wild-type strain thatcorresponds to the immunizing serotype(s). If wild-type virus infectiondoes occur, the host is resistant to developing moderate or severerotaviral disease, particularly of the gastrointestinal tract.

The multivalent immunogenic compositions of the present inventioncontaining the human×rhesus reassortant rotaviruses and rhesus rotavirusare administered to an infant, particularly a neonate, susceptible to orotherwise at risk of rotavirus disease to induce the individual's ownimmune response capabilities. Such an amount is defined to be an“immunogenically effective dose.” Immunogenicity or “immunogenicallyeffective dose” as used in the present invention means the developmentin a vaccinee of a cellular and/or antibody mediated immune response tothe vaccine composition. Usually such a response consists of thevaccinee producing serum antibodies, B cells, helper T cells, suppressorT cells, and/or cytotoxic T cells directed specifically to an antigen orantigens included in the vaccine composition of the present invention. Afour-fold or greater rise above a preinoculation antibody titerfollowing immunization measured by a rotavirus group specific, orrotavirus serotype-specific assay is considered a significant response.

In this method, the precise amount of each human×rhesus reassortantrotaviral serotype and rhesus rots virus in a particular immunogeniccomposition depends on the patient's age, state of health and weight,the mode of administration, the nature of the formulation, etc., butgenerally the range was from about 10⁴ to about 10⁶ plaque formingunits, preferably from about 10⁵ to less than 10⁶ plaque forming units(pfu) of each serotype per patient.

In any event, the immunogenic composition should provide a quantity ofeach reassortant rotavirus sufficient to induce an individual's immuneresponse against rotavirus disease. Preferably, this immune responsewill effectively protect the individual against serious orlife-threatening rotavirus disease. In some instances it may beadvantageous to combine the rotaviral compositions used in the methodsof the present invention with other serotypes of human rotavirus orother infectious agents, particularly, other gastrointestinal viruses.For example, the rotaviral compositions used herein can further include,for example, human×rhesus reassortant rotavirus of serotype 5(Timenetsky et al., J. General Virol. 78:1373-1378 (1997)), and/orserotype 9 (Nakagomi et al., Microbiol. Immunol. 34:77-82 (1990)),and/or serotype 10, and/or human×rhesus reassortant rotavirus of VP4serotype 1A. Administration can be simultaneous (but typicallyseparately) or sequentially with another possible gastrointestinal virusvaccine, such as a human calicivirus (e.g., Norwalk virus) or relatedvaccine.

In other embodiments, the invention provides for administration to aneonate an immunogenically sufficient amount of a multivalenthuman×bovine reassortant rotavirus composition comprising at least fourVP7 serotypes of human rotavirus. In one embodiment the human×bovinereassortant rotavirus which comprise the composition include a humanrotavirus serotype 1×bovine rotavirus strain UK, a human rotavirusserotype 2×bovine rotavirus strain UK, a human rotavirus serotype3×bovine rotavirus strain UK, and a human rotavirus serotype 4×bovinerotavirus strain UK. The multivalent composition can also include, butis not limited to, i.e., a human×bovine reassortant rotavirus ofserotype 5, and/or serotype 9, or a bovine×bovine reassortant rotaviruswith human rotavirus VP7 serotype 10 specificity, or a human rotavirusserotype VP4 1A×bovine rotavirus UK reassortant and the like. Further,as additional rotavirus serotypes are recognized as important in humandisease, they too can be added to an immunogenic composition of thepresent invention and used in methods for stimulating the immune systemto produce an immunogenic response to currently recognized and newlyrecognized rotaviruses of clinical significance.

Single or multiple administrations of the immunogenic compositions ofthe invention can be carried out. In neonates and infants, multipleadministrations may be required to elicit a sufficient level ofimmunity, particularly where there are high levels of maternally derivedantibodies specific for rotavirus. Administration should begin withinthe first 7 to 10 days of life, and continue at intervals such as one totwo months or more after the initial immunization, or as necessary toinduce and maintain sufficient levels of immunity against humanrotavirus infection. Levels of induced immunity can be monitored bymeasuring amounts of rotavirus group-specific antibodies orserotype-specific neutralizing antibodies in serum and secretions, anddosages adjusted or vaccinations repeated with one or more serotypes ofa multivalent reassortant rotavirus composition of the present inventionwhen necessary to maintain desired levels of immunity.

Thus, the methods of the invention specifically comprise a theadministration of an immunogenically sufficient amount of an immunogenicrotavirus composition to a neonate in need of immunological protectionagainst rotavirus followed by a second dose at about 2 to 4 months, andoptionally a third dose at 4 to six, or even 8 to 12 months of age.Administration of the first dose prior to the tenth day of life has beendemonstrated to prevent the transient febrile condition observed whenthe composition is initially administered at 2 to 6 months of age.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE

This example describes the administration of live oral rhesustetravalent (RRV-TV) vaccine in a double-blind placebo-controlled trialto compare the reactogenicity and immunogenicity of the composition whengiven at approximately 0, 2, and 4 months; 0, 4 and 6 months; or 2, 4,and 6 months. Placebo was administered at approximately 6 months to thefirst group, at 2 months for the second group and at 0 months for thethird group.

Most of the clinical procedures for the pediatric studies were alsoidentical to those described by Halsey et al., J. Infect. Dis.158:1261-1267 (1988)(incorporated herein by reference), with a fewexceptions. Briefly, routine childhood immunizations appropriate for thechild's age were given on schedule. Each pediatric subject wasrandomized to receive rotavirus or placebo. Buffered diluent(sodium-bicarbonate; citric acid) was used for reconstituting thelyophilized vaccine after which 2.5 ml of the mixture was withdrawn intothe dispette and administered orally. There were no feeding restrictionsbefore or after vaccination.

Rectal temperatures were taken daily and symptoms, if any, were recordedfor 7 days. Study subjects were considered to have “rotavirus-likeillness,” (i.e., an illness that could possibly be caused by arotavirus, if they had diarrhea, or any episode of frank vomiting orfever during the 7-day period after oral administration of rotavirus.Diarrhea was defined as three or more unformed stools within 48 hours.Fever was defined as a rectal temperature >38° C.

Blood was collected from each study participant before vaccination (cordblood); approximately one month after the second and third dose of“vaccine” (i.e., at approximately 5 and 7 months of age); and 4-6 weeksafter administration of rotavirus for measurement of rotavirus-specificantibodies. Prevaccination and postvaccination sera were tested forrotavirus-specific IgA and IgG antibodies by ELISA, using rhesusrotavirus as a group-specific antigen as described in Midthun et al., J.Clin. Microbiol. 27:2799-2804 (1989) and Hoshino et al., J. Clin.Microbiol. 21:425-430 (1985); each incorporated by reference herein. The3 sera were also tested by plaque reduction neutralization (PRN)antibody assay as described in Midthun et al., J. Clin. Microbiol.27:2799-2804 (1989). A fourfold or greater rise in antibody titer in thepostvaccination serum compared to the prevaccination serum measured byELISA IgA or PRN antibody assay was considered a significant response.

The rates of illness of vaccinees and placebo recipients and the ratesof serologic response for these groups within each age group and in eachstudy are compared using a two-tailed Fisher's exact test.

None of the 62 infants, who received RRV-TV vaccine at neonatal age, hadfever. Of the neonatally vaccinated infants, 0 of 32 who received asecond dose at 2 months of age, had fever as compared with thisindicates that neonatal immunization provided a level of protectionsufficient to prevent adverse reaction to the second dose of vaccineadministered at the time (i.e., 2 months of age) at which vaccinereactogenicity is characteristically expressed. This level of resistanceto rotavirus should serve as a foundation for a significant boosterresponse to the second and third doses of vaccine. Seroconversion byrhesus rotavirus neutralizing antibodies occurred in 71%, 86% and 89%and rotavirus IgA antibodies were detected in 68%, 87%, and 96% of theinfants after immunization schedules 0-2-4; 0-4-6 and 2-4-6 months,respectively.

Despite what appears to be lower immunogenicity, administration ofRRV-TV vaccine in the neonatal period is an alternative to thepreviously recommended 2-4-6 month schedule because of lowerreactogenicity and thus apparently greater safety. In addition, byadministering the first dose in the neonatal age, advantage is taken ofthe relatively refractory period during the first two months of life forthe development of intussusception (Rennels et al., Ped Infant10:924-925 (1998); Gay et al., Lancet 354:956 (1999)). Also, the absenceof fever after the second dose of vaccine at 2 months of age may be asignal of restricted multiplication of the vaccine during this criticalperiod when the incidence of intussusception begins its ascent (Rennelset al., ibid.; Fay et al., ibid). Neonatal immunization with the use ofbovine rotavirus based vaccine, already known to be more attenuated thanrhesus rotavirus but just as immunogenic, should provide a considerableincrease in safety reassured by not only a lack of reactogenicity butalso significant reduction in vaccine induced intussusception.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A method for reducing the incidence of a transient low level feverand/or intussusception associated with administration of a live rhesusrotavirus immunogenic composition to an infant which comprisesadministering the live rhesus rotavirus immunogenic composition to theinfant within about the first 7 to about 10 days of life followed by atleast one booster immunization prior to 6 months of age.
 2. The methodaccording to claim 1, wherein the composition comprises a rhesusrotavirus immunologically cross reactive with human rotavirus serotype3.
 3. The method according to claim 1, wherein the composition comprisesa human x rhesus reassortant rotavirus.
 4. The method according to claim3, wherein the composition comprises multiple human x rhesus reassortantrotaviruses of different serotypes.
 5. The method according to claim 4,wherein the composition comprises multiple human x rhesus reassortantrotaviruses of different serotypes and a rhesus rotavirusimmunologically cross reactive with human rotavirus serotype
 3. 6. Themethod according to claim 5, wherein the composition comprises a human xrhesus reassortant rotavirus of human rotavirus serotype 1, humanrotavirus serotype 2 and human rotavirus serotype 4 and a rhesusrotavirus immunologically cross reactive with human rotavirus serotype3.
 7. The method according to claim 4, wherein each rotavirus of a humanrotavirus serotype is administered separately.
 8. The method accordingto claim 4, wherein each rotavirus of a human rotavirus serotype isadministered in a combined composition.
 9. The method according to claim1, wherein the composition is administered to the alimentary tract of anindividual.
 10. The method according to claim 9, wherein the compositionis administered as a liquid suspension.
 11. The method according toclaim 6, wherein the booster is administered at about 6 to about 10weeks of age.
 12. The method according to claim 11, wherein a furtherbooster is administered at about 14 to about 18 weeks of age.
 13. Themethod according to claim 6, wherein the booster is administered atabout 14 to about 18 weeks of age.
 14. The method according to claim 13,wherein a further booster is administered at about 22 to about 26 weeksof age.